The main goals of this Program Project are to: 1) understand the molecular and cellular mechanisms by which the integral membrane enzyme fatty acid amide hydrolase (FAAH) degrades neuromodulatory fatty acid amides (FAAs), and 2) determine the physiological and potential pathological consequences of chemically inhibiting FAAH in vivo. FAAs represent an emerging class of lipid messengers that influence a variety of behavioral processes, including, pain sensation, anxiety, sleep, and feeding. Recent genetic and pharmacological studies have demonstrated that FAAH is a principal regulator of FAA-based signaling events in vivo, suggesting that this enzyme may represent an attractive target for the treatment of neurological disorders like pain and anxiety. Nonetheless, many questions remain regarding the mechanism of action of FAAH and its suitability as a therapeutic target. For example, among the numerous hydrolases present in mammalian proteomes, how does FAAH exert such extraordinary control over the levels and activity of FAAs? Might FAAH recruit its FAA substrates directly from cell membranes in order to expedite their inactivation? Can potent and selective reversible inhibitors of FAAH be generated that, upon pharmacological administration, reproduce the neurochemical and behavioral effects observed in FAAH-knockout mice? It is the goal of this application to address these important questions by embarking on a multidisciplinary program aimed at: 1) testing the function of unique domains in FAAH, including its provocative collection of channels and membrane-binding sites (Project I), 2) determining the crystal structures of key FAAH species, including the apo-enzyme, inhibitor/product complexes, and mutants like the natural P129T variant associated with problem drug use (Project II), and 3) evaluating the neurochemical and behavioral effects of FAAH inhibitors in vivo (Project III). The knowledge gained from these studies will also be applied towards the design of increasingly potent and selective FAAH inhibitors (Core), which should prove of great value as both research tools and potential therapeutic agents.